Exchange-induced remnant magnetization for label-free detection of dna, micro-rna, and dna/rna-binding biomarkers

ABSTRACT

A method of using an exchange-induced remnant magnetization (EXIRM) technique for label free detection of short strands of nucleotides and cancer biomarkers, such as DNA and microRNA strands, DNA/RNA-binding biomarkers, and cancer-specific antigens, with high sensitivity, high specificity, and broad dynamic range. The method may provide a label-free approach aimed to facilitate high reliability, and to require a minimum amount of biochemical reagents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/247,953, filed Apr. 8, 2014, which claims priority to U.S.Provisional Patent Application No. 61/810,575, filed Apr. 10, 2013, thecontent of each being hereby incorporated by reference in their entiretyfor all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant no.ECCS-1028328 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

BACKGROUND Field of the Invention

The present disclosure relates to the fields of miRNA profiling andbiomarker detection.

Background

Nucleotides are biological molecules that form the building blocks ofnucleic acids (DNA and RNA) and serve to carry packets of energy withinthe cell (ATP). In the form of the nucleoside triphosphates (ATP, GTP,CTP and UTP), nucleotides play central roles in metabolism. In addition,nucleotides participate in cell signaling (cGMP and cAMP), and areincorporated into important cofactors of enzymatic reactions (e.g.coenzyme A, FAD, FMN, NAD, and NADP⁺). Nucleotides may also comprisesynthetic sequences, and comprise chemical modifications to thenucleotide structure to produce for example nucleotide analogues.

DNA and RNA are biological molecules that are essential to life. Geneticinformation is encoded as specific sequences of DNA molecules. Theinformation is passed along during transcription and protein synthesisvia messenger RNA. Therefore, they are closely related, and interplaywith various types of diseases, such as cancers.

In particular, miRNAs play a significant role in gene regulation, andare consequently a major category of biomarkers for cancer diagnostics.Because of their short strands and diverse expression levels, it remainstechnically challenging to achieve precise and quantitative detection.MiRNAs which are short RNA strands containing 18-25 nucleotides, playnumerous important roles, including those in gene expression,development, and cell differentiation (1-3). The mature miRNAsincorporate into RNA-induced silencing complexes that bind withmessenger RNAs based on partial sequence complementarity andconsequently cause inhibition of protein translation. The regulation bymiRNAs depends on their sequence, expression level, and cooperation withother miRNAs. Therefore, sensitive and specific detection of miRNAs isan essential step towards understanding their roles in proteinsynthesis, cell death, and as biomarkers of disease.

A range of techniques have been used for miRNA profiling. Known methodsinclude northern blotting (4), reverse transcriptase polymerase chainreaction (5), in situ hybridization (6), microarray (7), bioluminescence(8), surface plasmon resonance (9), surface-enhanced Raman spectroscopy(10), electrochemical detection (11), fluorescence (12), and photonicmethods (13); however, no single technique achieves high sensitivity,single-base specificity, and broad dynamic range. In addition,reproducibility remains a significant issue when comparing results fromdifferent techniques, due to the many steps and various protocolsinvolved in analysis (14). Hence there is an unmet need in the field fora single technique that can detect short nucleotide sequences such asmiRNA, with high sensitivity, high specificity, and broad dynamic range,which may be a one-step method that facilitates high reliability, andneeds minimum amount of biochemical reagents.

Thus, the production of a method capable of accurately detecting shortnucleotide sequences, (DNA or RNA sequences such as miRNA) with highsensitivity, high specificity, and broad dynamic range would beparticularly well received, and embodiments of the herein presentedmethod are believed to overcome certain above mentioned limitations bythe utilization an exchange-induced remnant magnetization (EXIRM)technique (15).

In addition, many cancer biomarkers can specifically bind with short DNAstrands, for example prostate specific antigen (16). The EXIRM methodcan be directly modified to achieve sensitive and label-free detectionof such cancer biomarkers.

BRIEF SUMMARY OF THE DISCLOSED EMBODIMENTS

The present disclosure relates to a method of using an exchange-inducedremnant magnetization (EXIRM) technique for detecting short strands ofnucleotides, such as those comprising deoxyribonucleic acid (DNA); andthose comprising ribonucleic acid (RNA), including microRNA (miRNA),with high sensitivity, high specificity, and broad dynamic range.Further, the method herein described may also be a one-step method thatfacilitates high reliability, and requires a minimum amount ofbiochemical reagents.

Certain embodiments herein described address such needs, and usessequence-specific exchange reactions between label-free nucleotidesequences (for example miRNA) and magnetically labelled nucleotidesequences (for example RNA, miRNA, or DNA) with, in some embodiments aone base difference. In one embodiment, the exchange-induced remnantmagnetization (EXIRM) quantitatively measures a target miRNA withsingle-base specificity, and in some embodiments the detection limit ofsuch target miRNAs reach zeptomolar levels. In a further embodiment, twomiRNAs with only one base difference may be detected in parallel whileshowing no magnetic signal cross-talking, and in still furtherembodiments, the EXIRM technique analyses miRNA without anyamplification or washing procedures. In some embodiments the EXIRMmethod herein described is suitable for precise miRNA profiling forearly diagnosis and precise prognosis of cancers. The method can also beextended, wherein some embodiments a sample of interest for which aquantitative measurement is required may comprise a protein or aderivative thereof, and in a further embodiment a sample may comprise anantibody. Further such measurements may also be performed directly in abiological environment such as but not limited to blood plasma, urine,or cell lysate, and or other environments with limited opticaldetection.

In one embodiment, a method of detecting nucleotide sequences comprises:(a) immobilizing a first nucleotide single strand on a surface; (b)adding a second nucleotide single strand to the first nucleotide singlestrand to form a hybridized double strand, where the second strandcomprises a first magnetic particle; and a nucleotide sequence that isless than 100% complementary to the first nucleotide single strand, andcomprises at least a first mismatched base; (c) measuring a firstmagnetic signal value for the hybridized double strand; (d) incubating athird nucleotide strand with the hybridized double strand; wherein thethird strand is complementary to the first strand, and whereinincubating forms an exchange product; (e) measuring a second magneticsignal value for the exchange product of step d; and (f) quantifying theamount of the third nucleotide strand from the difference in magneticsignal values measured in step c and step e.

In some embodiments of the method of detecting nucleotide sequences, thefirst nucleotide single strand is derivatized; in some other embodimentsthe first nucleotide strand may be biotinylated or thiol captured. Inanother embodiment of the method of detecting nucleotide sequences thefirst nucleotide strand is immobilized to a surface through a S—Aucovalent bond. In a further embodiment of the method of detectingnucleotide sequences, a magnetic particle is attached to the secondnucleotide strand by a streptavidin-biotin covalent bond. In anotherembodiment the magnetic particle is about 1 nm to about 10 μm in size(for example, diameter of spherical magnetic particles). in a furtherembodiment about 10 nm to about 5 μm in size, and in a furtherembodiment the magnetic particle is about 3 μm in size.

In another embodiment of the method of detecting nucleotide sequences,measuring comprises an atomic magnetometer; in a further embodiment, thefirst and the second magnetic signal values comprise magnetic momentmeasurements (17), and in another embodiment of the method of detectingnucleotide sequences, step (f) comprises measuring the change inmagnetic signal (ΔB), and in some further embodiments the molarconcentration of the third nucleotide strand may be calculated whereinthe molar concentration is linearly related to ΔB or the change inmagnetic moment measurements. In some embodiments of the method ofdetecting nucleotide sequences, quantifying further comprisescalculating the number of free magnetic particle labels, wherein thenumber of said free magnetic particles corresponds to the number ofexchange product molecules.

In some embodiments of the method of detecting nucleotide sequences, thesurface is in a sample holder. In one embodiment, the hybridized doublestranded sequence is in a liquid environment. In further embodiment theenvironment is a cell lysate, and in a still further embodiment theenvironment is blood plasma, and in a further embodiment, theenvironment is urine.

In some embodiments of the method of detecting nucleotide sequences, thefirst nucleotide strand is a RNA or a DNA sequence, in anotherembodiment the third nucleotide strand is a DNA or microRNA sequence. Insome embodiments, the first nucleotide strand is about 1-100 nucleotidesin length, in some further embodiments, the second nucleotide strand isabout 1-100 nucleotides in length, and in some still further embodimentsthe third nucleotide strand is about 1-100 nucleotides in length.

In some embodiments, the first nucleotide strand is about 10-50nucleotides in length, in some further embodiments, the secondnucleotide strand is about 10-50 nucleotides in length, and in somestill further embodiments the third nucleotide strand is about 10-50nucleotides in length.

In some embodiments, the first nucleotide strand is about 18-25nucleotides in length, in some further embodiments, the secondnucleotide strand is about 18-25 nucleotides in length, and in somestill further embodiments the third nucleotide strand is about 18-25nucleotides in length.

In some embodiments, the first nucleotide strand is about 18-25nucleotides in length, in some further embodiments, the secondnucleotide strand is replaced by the DNA/RNA-binding biomarker, and insome still further embodiments the third nucleotide strand is about18-25 nucleotides in length.

In other embodiments of the method of detecting nucleotide sequences,the exchange product is thermodynamically more stable than thehybridized double strand, in some embodiments the double strand is 12 pN(pN: 10⁻¹²N) less stable than said exchange product.

In another embodiment, a method of simultaneously detecting an array ofheterologous nucleotide sequences is provided wherein the methodcomprises: (a) coating a sample well comprising an array of compartment;wherein the surface of adjacent compartments are alternatively coatedwith i) a hybridized nucleotide double strand; and ii) are uncoated;wherein the uncoated compartment produces no magnetic signal; and eachcoated compartment comprises a heterologous hybridized double strandsequence; (b) measuring magnetic signals for each compartment; (c)incubating the array with a sample comprising free target nucleotidesequences, and forming exchange products; (d) measuring magnetic signalsfor each compartment comprising exchange products after applying a weakmechanical force to remove nonspecifically bound magnetic particles; (e)calculating the difference in said signals from step b and d; and (f)quantifying and identifying said target sequence based on the change insignal calculated in step (e). In a further embodiment of the method ofsimultaneously detecting an array of heterologous nucleotide sequencesmeasuring the magnetic signal from the sample array is by: a scanningsingle sensor, scanning the sample well, a two-dimensional sensor arrayfor simultaneous detection or combinations thereof.

In another embodiment, an exchange induced remnant magnetization methodto detect specific nucleotide sequences is herein described, the methodcomprising: (a) immobilizing a first single stranded sequence on asurface; wherein the first sequence comprises N bases; (b) adding asecond single stranded sequence to the first single stranded sequence,wherein the second single stranded sequence comprises N−1 complementarybases; wherein said complementary bases are complementary to thesequence of the first single strand sequence, and wherein the secondsingle stranded sequence hybridizes to the first single strandedsequence forming a hybridized double stranded sequence with N−1 basepairs; and (c) incubating the hybridized double stranded sequence with athird single stranded sequence, wherein the third single strandedsequence comprises N complementary bases, wherein said complementarybases are complementary to the sequence of the first single strandsequence; and wherein said third single stranded sequence exchanges withsaid second single stranded sequence to form an exchange productcomprising a double strand with N complementary base pairs; wherein saidexchange product is thermodynamically more stable than said hybridizeddouble stranded sequence. Further embodiments may include specieswherein the second single stranded sequence is mismatched by greaterthat one complementary base.

In another embodiment, an exchange induced remnant magnetization methodto detect specific biomarkers is herein described, the methodcomprising: (a) immobilizing a first single stranded sequence on asurface; wherein the first sequence comprises N bases; (b) adding asecond single stranded sequence to the first single stranded sequence,wherein the second single stranded sequence comprises N complementarybases; wherein said complementary bases are complementary to thesequence of the first single strand sequence, and wherein the secondsingle stranded sequence hybridizes to the first single strandedsequence forming a hybridized double stranded sequence with N basepairs; and (c) incubating the hybridized double stranded sequence with abiomarker, wherein the biomarker exchanges with said second singlestranded sequence to form an exchange product comprising a DNA-biomarkercomplex; wherein said exchange product is thermodynamically more stablethan said hybridized double stranded sequence. Further embodiments mayinclude species wherein the second single stranded sequence ismismatched by one complementary base or more.

Thus, embodiments described herein comprise a combination of featuresand characteristics intended to address various shortcomings associatedwith certain methods of detecting nucleotide sequences such as DNA andmicroRNA sequences, wherein the exchange-induced remnant magnetizationtechnique described detects such sequences with high sensitivity, highspecificity, and over a broad dynamic range as compared to sometechniques known in the art. The various features and characteristicsdescribed above, as well as others, will be readily apparent to thoseskilled in the art upon reading the following detailed description, andby referring to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For a detailed description of the disclosed embodiments, reference willnow be made to the accompanying drawings, wherein:

FIG. 1 depicts an embodiment of the EXIRM technique described herein,where three RNA strands are involved: immobilized Strand 1, hybridizedStrand 2 with one mismatching base, and Strand 3 which is the targetmiRNA. Strand 2 is labelled by magnetic particles (the approach may alsobe applied to DNA);

FIG. 2 depicts a plot of a magnetic signal changes (ΔB (in pT; 10⁻¹² T))for various concentrations (femto molar, 10⁻¹⁵ M) of the target DNA,wherein the linear correlation shows good quantification of the DNAmolecules in accordance with principles described herein. The diameterof the magnetic particles is 2.8 μm in accordance with principlesdescribed herein;

FIG. 3 depicts a graphic representation of normalized measurements with1 second signal averaging for the 12-base DNA exchange reaction at fivedifferent concentrations; wherein the normalized profiles were obtainedin this embodiment by averaging 33 adjacent data points in the raw data,where each data point was measured for 30 ms, the X-axis represents therelative position of the sample to the atomic sensor during scanning thesample, and performed in accordance with principles described herein;

FIG. 4 depicts a plot of magneto-optical resonance to show the maximummeasurable magnetic fields by the atomic magnetometer, wherein thehighlighted rectangular area shows the responsive range of the atomicmagnetometer to the samples magnetic signal, in accordance withprinciples described herein;

FIG. 5 depicts a plot of a magnetic signal changes ΔB (pT) for variousconcentrations of the target DNA using smaller magnetic particles, withdiameter of 1 μm, and shows the quantitative relationship between thechange in B with concentration of magnetic particle labelled DNA;

FIG. 6 depicts an illustration of multiplexed detection, in accordancewith principles described herein and wherein (A) is a Schematic of anarray of sample wells. Each golden square is for a specific target ofRNA or DNA. Each blank separates two adjacent sample wells; and (B) isan example of detecting multiple targeted RNA or DNA. The atomicmagnetometer is composed of an atomic sensor housed in a multi-layeredmagnetic shield, the sample cell containing an array of sample wells isintroduced through a sample inlet channel through the magnetic shield,and an optical fiber may be used to deliver the laser beam to the atomicsensor. The signal comes out through a signal cable;

FIG. 7 depicts a plot of a magnetic signal as a function of reactiontime for an EXIRM experiment; wherein a 12-base DNA was the targetstrand, which replaced another DNA with one-base difference that waspre-hybridized with the complementary strand to the former (in control,the target DNA was absent) performed in accordance with principlesdescribed herein;

FIG. 8 (A-E) depicts EXIRM for multiplexed miRNA analysis withsingle-base specificity; (A) shows the MiRNA sequences let-7a, 1a; 2;1b; 2; let-7c respectively; which are used in the correspondingexperiment; and bases that are used for the analysis are in red (fourthline) with the mismatching bases underlined; (B) shows an imagedepicting a sample holder with two sample wells; (C); (D); and (E)depict the magnetic signal change for adding let-7c (C), let-7a (D), andno miRNA (E) into both of the sample wells shown in B; The dashed linesin C, D, and E, show the positions of the two sample wells.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

Embodiments herein addressed are intended to overcome certain abovementioned limitations by using a method for an exchange-induced remnantmagnetization (EXIRM) technique for detecting DNA, microRNA, andDNA/RNA-binding proteins with high sensitivity, high specificity, andbroad dynamic range (15). Herein and throughout the application, theterm “strand” and “sequence” may be used interchangeably to describesequences of nucleotides which are single stranded. Similarly,“hybridized strand,” “hybridized double strand,” “hybridized doublestranded sequence” or “hybridized sequence” may be used interchangeably.As used herein, the term “about,” when used in conjunction with apercentage or other numerical amount, means plus or minus 10% of thatpercentage or other numerical amount. For example, the term “about 80%,”would encompass 80% plus or minus 8%. Further, all references citedherein are incorporated in their entirety.

General Principle

In some embodiments, an exchange induced remnant magnetization method todetect specific nucleotide sequences is herein described, the methodcomprises: (a) immobilizing a first single stranded sequence on asurface; wherein the first sequence comprises N bases; (b) adding asecond single stranded sequence to the first single stranded sequence,wherein the second single stranded sequence comprises N−1 complementarybases; wherein said complementary bases are complementary to thesequence of the first single strand sequence, and wherein the secondsingle stranded sequence hybridizes to the first single strandedsequence forming a hybridized double stranded sequence with N−1 basepairs; and (c) incubating the hybridized double stranded sequence with athird single stranded sequence, wherein the third single strandedsequence comprises N complementary bases; and wherein said third singlestranded sequence exchanges with said second single stranded sequence toform an exchange product comprising a double strand with N complementarybase pairs; wherein said exchange product is thermodynamically morestable than said hybridized double stranded sequence.

In some embodiments, a method of detecting nucleotide sequences isherein described, comprising (a) immobilizing a first nucleotide singlestrand on a surface; (b) adding a second nucleotide single strand to thefirst nucleotide single strand to form a hybridized double strand, wherethe second strand comprises a first magnetic particle; and a nucleotidesequence that is less than 100% complementary to the first nucleotidesingle stand, and comprises at least a first mismatched base; (c)measuring a first magnetic signal value for the hybridized doublestrand; (d) incubating a third nucleotide strand with the hybridizeddouble strand; wherein the third strand is complementary to the firststrand, and wherein incubating forms an exchange product; (e) measuringa second magnetic signal value for the exchange product of step d afterapplying a weak mechanical force to remove nonspecifically boundmagnetic particles; and (f) quantifying the amount of the thirdnucleotide strand from the difference in magnetization value measured instep c and step e.

Thus, in one embodiment of the invention herein described, a specificnucleotide sequence such as (but not limited to) a miRNA sequence can bedetected by magnetic signal changes caused by exchange reactions betweena target miRNA sequence and a magnetically labelled RNA sequence with asingle nucleotide base difference; as illustrated in the embodiment ofFIG. 1, a hybridized RNA double strand is first prepared, a strandcomposed of nucleotide bases that compliments the target miRNA(Strand 1) is immobilized on the surface of a sample well/plate, and asecond strand (Strand 2) composed of nucleotide bases that differ (aremismatched) by (at least) one base from Strand 1, and the target miRNA(Strand 3) is further labelled with a magnetic particle. Strand 1 andStrand 2 form a hybridized double strand of RNA, immobilized on thesurface through Strand 1 and magnetically labelled through Strand 2.

The target miRNA is then incubated with the hybridized double strand inthe sample well. An exchange reaction then takes place, in which thetarget miRNA replaces the mismatching strand because the former hasthermodynamically stronger binding with the immobilized RNA than thelatter. When the strands with one mismatching base (hybridized pair)which are immobilized and magnetically labelled undergo magnetization bya strong magnet (>0.1 Tesla), the magnetic dipoles of the particles arealigned and produce a strong magnetic signal; hence when the mismatchingRNA undergoes dissociation from the immobilized strand due to thethermodynamically favoured binding of the complementary target strand,randomization of the magnetic dipoles of the magnetic labels occurs dueto Brownian motion of the now free magnetically labelled strands whichis induced by a weak mechanical force provided by a shaker or acentrifuge. The exchange reaction thus produces a decrease in themagnetic signal (ΔB in pT), because of the randomization of the magneticparticles, which is measured by an atomic magnetometer (17), thedecreasing amplitude of the signal thus represents the quantity of thetarget miRNA molecules.

Thus, in some embodiments of the method of detecting nucleotidesequences, the first nucleotide strand is a RNA or a DNA sequence, inanother embodiment the third nucleotide strand is a DNA or microRNAsequence. In some embodiments, the first nucleotide strand is about1-100 nucleotides in length, in some further embodiments, the secondnucleotide strand is about 1-100 nucleotides in length, and in somestill further embodiments the third nucleotide strand is about 1-100nucleotides in length.

In some embodiments, the first nucleotide strand is about 10-50nucleotides in length, in some further embodiments, the secondnucleotide strand is about 10-50 nucleotides in length, and in somestill further embodiments the third nucleotide strand is about 10-50nucleotides in length. In some embodiments, the first nucleotide strandis about 18-25 nucleotides in length, in some further embodiments, thesecond nucleotide strand is about 18-25 nucleotides in length, and insome still further embodiments the third nucleotide strand is about18-25 nucleotides in length. In some embodiments the second nucleotidesingle strand comprises 1-100 mismatched bases, in another embodimentthe second nucleotide single strand comprises 1-50 mismatched bases, inanother embodiment the second nucleotide single strand comprises 1-10mismatched bases; and in a preferred embodiment the second nucleotidesingle strand comprises 1 mismatched based, wherein the definition ofmismatched is that the base bonds with a second molecule or base that isnot it's natural Watson and Crick base pair interaction i.e.guanine/cytosine bonding, adenine/thymine bonding and adenine/uracilbonding.

In other embodiments of the method of detecting nucleotide sequences,the exchange product is thermodynamically more stable than saidhybridized double strand, in some embodiments the double strand is atleast 12 pN less stable than said exchange product, wherein thestability declines based on the loss of hydrogen bonding between basepairs.

In some embodiments of the method of detecting cancer biomarkers, theexchange product of the DNA-biomarker complex is thermodynamically morestable than said hybridized double strand.

In some embodiments of the method of detecting nucleotide sequences, thefirst nucleotide single strand is derivatized; in some other embodimentsthe first nucleotide strand may be biotinylated or thiol captured. Inanother embodiment of the method of detecting nucleotide sequences, thefirst nucleotide strand is immobilized to a surface through a S—Aucovalent bond. In a further embodiment of the method of detectingnucleotide sequences, a magnetic particle is attached to the secondnucleotide strand by a streptavidin-biotin covalent bond. In anotherembodiment the magnetic particle is about 1 nm to about 10 μm in size,about 10 nm to about 5 μm in size, and in a further embodiment themagnetic particle is about 3 μm in size.

Detection Level and Sensitivity of the Method

The level of detection for the embodiments of the method hereindescribed is obtained by varying the concentration of the targetnucleotide sequences, for example the magnetic signal decrease (ΔB) isplotted against the concentration of target nucleotides, in one suchembodiment illustrated in FIG. 2, five different concentrations of a DNAtarget sequence were used, and EXIRM performed. The five measurements ofΔB were linearly correlated, and the linear fit goes through the origin,which indicates the high sensitivity of the method for measuring changesin magnetic signal at these concentrations, and that the quantificationis highly reliable.

In some embodiments, given a sample well with a known volume of 8 μL,the total number of the DNA sequences being replaced can be calculatedto be 660 zeptomole, or 4×10⁵ molecules, for the concentration of 83 fM.The error bars were obtained by normalizing the measuring time to 1second, which was nearly 1 pT (FIG. 3). From the ΔB value of 12 pT forthe 83 fM sample, the detection limit has already reached 3.3×10⁴molecules. Therefore, the detection limit is substantially better thanother current techniques for DNA/RNA oligomers (18). In a furtherembodiment, based on the sensitivity of 150 fT of the apparatus (19),the detection level of the method can allow measurements of 6×10³molecules with 1 second signal averaging time.

Dynamic Range

The dynamic range of EXIRM is defined as the span between the number oftarget molecules that give the lowest detectable magnetic signal and thenumber of target molecules that give the highest detectable magneticsignal.

The dynamic range of embodiments of the method described herein, can bederived from the sensitivity of the magnetometer and the width ofmagneto-optical resonance. The latter represents the optical response bythe magnetometer to the magnetic field to be measured. Therefore, itprovides the range for the magnetic signal that the magnetometer issensitive to. In some embodiments, a sensitivity of about 150 fT definesthe lower end of the dynamic range. In some embodiments, given aresonance width of ˜70 Hz for the magnetometer (FIG. 4, highlightedarea) a frequency range (the horizontal axis) is the frequency change isalmost linear to the magnetic signal. This width gives an uppermeasurement limit of about 10 nT, because B=ω/2γ, where ω is themodulation frequency of the laser and γ of 3.5 Hz/nT is the gyromagneticratio for Cs (cesium, the atom for the atomic magnetometer shown here).Hence in one such embodiment, the dynamic range is about 5 orders ofmagnitude for a selected magnetic particle.

In another embodiment, a broad dynamic range is preferred for miRNAprofiling, because it is well known that the expression levels may bedrastically different for different miRNAs. Therefore in someembodiment's large numbers of miRNA are available for exchange whereinthe signal change will be greater, while in other embodiments the numberof miRNA expressed and available for exchange will be small. In oneembodiment of the method herein described, the atomic magnetometer hasan upper detection limit of approximately 10 nT; and the lower limit isdetermined by the sensitivity, which is 150 fT. Therefore, with aselected type of magnetic particles for labelling, the dynamic range isabout five orders of magnitude as described above.

Furthermore, in some embodiments, the dynamic range in terms of numberof miRNA molecules can be adjusted by tuning the magnetic property ofthe particles. This is because for magnetically weaker particles, alarger number of particles will be needed to reach the upper limit ofthe detection range. Hence more target molecules can be detected. Whilefor magnetically stronger particles, a fewer number of particles willprovide sufficient magnetic signal so that a fewer number of targetmolecules will be detected. Potentially single-molecule detection isachievable when the magnetic particle gives sufficiently strong signal(20). Therefore, again in some embodiments the dynamic range of EXIRMmay be greater than five orders of magnitude. An example of usingdifferent sized magnetic particles to adjust the dynamic range is shownin FIG. 5.

Multiplexed Detection

In another embodiment, a method of simultaneously detecting an array ofheterologous nucleotide sequences is provided wherein the methodcomprises: (a) coating a sample well comprising an array of squares;wherein the surface of adjacent squares are alternatively coated with i)a hybridized nucleotide double strand; and ii) remain uncoated; whereinthe uncoated square produces no magnetic signal; and each coated squarecomprises a heterologous hybridized double strand sequence; (b)measuring magnetic signals for each square; (c) incubating the arraywith a sample comprising free target nucleotide sequences, and formingexchange products; (d) measuring magnetic signals for each squarecomprising exchange products; (e) calculating the difference in saidsignals from step b and d; and (f) quantifying and identifying saidtarget sequence based on the change in signal calculated in step (e). Ina further embodiment of the method of simultaneously detecting an arrayof heterologous nucleotide sequences measuring the magnetic signal fromthe sample array is by: a scanning single sensor, scanning the samplewell, a two-dimensional sensor array for simultaneous detection orcombinations thereof.

Thus, multiplexed detection is a simultaneous measurement or a method ofidentifying multiple species in a single experimental run. In someembodiments, multiplexed detection assays are experiments that endeavourto detect or to assay the state of all biomolecules of a given class(e.g., miRNAs) within a biological sample, to determine the effect of anexperimental treatment or the effect of a DNA mutation over all of thebiomolecules or pathways in the sample.

In some embodiments of the method herein described, multiplexeddetection of arrayed samples is needed because miRNAs often do notfunction alone. It has been reported that groups of miRNAs playimportant roles cooperatively (21). In addition, miRNA expression ishighly heterogeneous (22). Therefore, monitoring a group of miRNAs thatmay be closely related in sequence in a single sample is required. FIG.6 shows an embodiment of a one-dimensional multiplexed detection. Thebottom of a single sample well is patterned into an array of squares,which are alternatively coated with RNA double strands which havesequences that vary cell to cell, and a blank. Each coated square aimsat targeting one type of miRNA sequence. In some embodiments, a blanksquare is needed in between two coated squares to avoid signalcross-talking that arises from the overlap of the magnetic signals. Inone embodiment, 2 mm atomic sensors are used, thus each sample square is2×2 mm², the distance between two adjacent samples is 4 mm center tocenter, and the area of the sample being 4×1 mm², thus in one suchembodiment, a sample well with 2.6×2.6 cm² bottom area can analyze 49different types of miRNAs. Smaller atomic sensors may be used formultiplexed detection of a greater number of different miRNAs if needed.

As such there are two ways to detect the magnetic signals from thesample array: one embodiment comprises a scanning single sensor, andanother embodiment comprises using a two-dimensional sensor array forsimultaneous detection. FIG. 6B shows a schematic of using the singlesensor approach. The approach of using an array of sensors can becarried out similarly.

In some embodiments, no amplification is needed, and no washing orsample transfer is used. This simplifies the analysis procedure andimproves the reliability of the measurements as compared to knowntechniques which often involve multiple steps of sample preparation,amplification, and multiple washing steps.

In some embodiments, the EXIRM technique described herein, provides anew avenue for miRNA analysis. In some embodiments, the high sensitivityof atomic magnetometers allows detection of about 10⁴ molecules; infurther embodiments detection may be in the order of 10³ molecules. Inother embodiments single-base specificity is achieved from thesequence-specific exchange reactions; and in further embodiments,cross-talking is not observed between miRNAs wherein in someembodiments, there is only a one base difference between sequences.

EXAMPLES Example 1(A) EXIRM's Single-Base Specificity in DNA Detection

In one embodiment, to demonstrate EXIRM, three 12-base DNA strands werechosen. The nucleotide strand was a thiolated GGG AAA AAA GGG (Strand1), which was loaded into a sample well (of 4×2×1 mm³ in size) andsubsequently immobilized on the bottom surface of the well via S—Aucovalent bonds (15).

The second strand was then added for hybridization, using biotinylatedoligonucleotide sequences, CCC AAA AAT CCC (Strand 2; 11 base pair matchto strand 1) and was labelled with magnetic particles (examples of suchmagnetic particles include Streptavidin-coated 2.8 μm sized magneticparticles (Invitrogen, M280)). The target strand (CCC AAA AAA CCC(Strand 3)) which was fully complimentary to the immobilized strand (12base pair match), was thus added to the sample well. This system waschosen because the force of Strand 1 binding to Strand 2 (11-base pairmatch) is 12 pN weaker than that of Strand 1 binding to Strand 3 (12base pair match). The magnetic signal showed a decrease when Strand 3was added into the sample well containing Strand 1-Strand 2 double helix(lower trace in FIG. 7). The decrease signal indicated the occurrence ofthe exchange reaction, in which Strand 3 replaced Strand 2. In contrast,when the target Strand 3 was absent, no magnetic signal decrease wasobserved (upper trace in FIG. 7). The magnetic signal profile alsoshowed that the exchange reaction took less than one hour to complete at37° C.

Example 1(B) EXIRM's Single-Base Specificity in Multiplexed MiRNAAnalysis

In one embodiment of the method herein described, two sample wells wereplaced in parallel along the sample holder (FIG. 8B), the two selectedtarget miRNAs were let-7a and let-7c, which are biomarkers for lungcancer (21) and colorectal cancer (22) respectively, and the two miRNAonly differ by one base as can be seen in FIG. 8A which shows theexperimental sequences. In this embodiment, nucleotide Strand 1a isentirely complimentary (base by base) with let-7a and nucleotide Strand1b is entirely complimentary (base by base match) with let-7c. The twosample wells used a common Strand 2, which had one mismatching base forboth Strand 1a and Strand 1b. When let-7c was added to both samplewells, only the right sample well (located at 182 mm) gave a magneticsignal decrease, as depicted by the magnetic signal change (ΔB(pT)) seenin FIG. 8C). This is because let-7c compliments Strand 1b and thereforereplaces Strand 2 only in the right sample well. Let-7c is one-basemismatched for Strand 1a, thus no exchange reaction occurred in the leftsample well (at 168 mm). Similarly, when let-7a was added to both samplewells, only the left sample well produced a magnetic signal decrease asdepicted by the magnetic signal change (ΔB(pT)) seen in FIG. 8D). As acontrol experiment, when no miRNA was added to the sample wells, nomagnetic signal change was observed in either sample well as depicted inFIG. 8E. This confirms the responses were due to the presence ofappropriate miRNAs as they exchanged with Strand 2 in the sample wellcontaining their respective complimentary strands, and that no crosstalking of magnetic signal is exhibited between let-7a and let-7c, thusthe EXIRM method herein described has single-base specificity. In someembodiments, this capability is required for miRNA profiling becausemiRNAs in the same family often differ by only a few nucleotides;conventional techniques are often unable to obtain single basespecificity especially in light of the short sequences often associatedwith miRNA's.

Materials and Methods.

In some embodiments, the biotinylated or thiol-derivitized firstnucleotide strands were immobilized on the bottom surface of samplewells wherein the surface is streptavidin- or gold-coated. Afterhybridization with their corresponding biotinylated strand 2, with atleast one mismatching base, the samples are incubated in on embodimentwith streptavidin-conjugated magnetic particles (Invitrogen M280) atroom temperature in tris-buffered saline (TBS) solution with 1% (w/v)bovine serum albumin (BSA) and 0.05% detergent Tween 20, the magneticparticles were then magnetized by a permanent magnet. The M280 particlesare uniform, superparamagnetic beads of 2.8 mm in diameter with astreptavidin monolayer covalently bound to the surface. They aresupplied as a suspension.

In some embodiments, to initiate the exchange reaction, the target DNAor miRNA with an entirely complementary sequence (to the capture probe)to strand, was then added and incubated in TE buffer (10 mM tris, 1 mMEDTA, 1 M NaCl, pH 8.0) at 37° C. The samples' magnetic signal wasmeasured by an atomic magnetometer after applying a weak centrifugalforce to eliminate physisorption of the magnetic particles. For the DNAexchange reaction, reaction time was varied between 20-220 min. FormiRNA targets (let-7a and let-7c), two capture probes (1a and 1b) werelocated in two sample wells were placed on a sample holder, one with thedouble helix of Strand 1a and Strand 2, and the other with Strand 1b andStrand 2. The center-to-center distance between the sample wells was 14mm. The reaction time was 6 hrs for miRNAs exchange.

In some embodiments, using miniature atomic magnetometers (23), EXIRM iscapable of sensitive and precise miRNA profiling, and in some furtherembodiments will be used in cancer diagnostics.

While certain embodiments of the invention described herein specificallyfocus on a novel method to detect DNA and miRNA sequences of interestbased on their specific binding pairs and specificity, one of ordinaryskills in the art, with the benefit of this disclosure, will recognizethe extension of the approach to other systems.

Other and further embodiments, versions and examples of the inventionmay be devised without departing from the basic scope thereof and thescope thereof is determined by the claims that follow. References citedherein are incorporated by reference in their entirety.

REFERENCES

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What is claimed is:
 1. A method of detecting nucleotide sequences,comprising a) immobilizing a first nucleotide single strand on asurface; b) adding a second nucleotide single strand to the firstnucleotide single strand to form a hybridized double strand, whereinsaid second strand comprises: a first magnetic particle; and anucleotide sequence that comprises at least a first mismatched base; c)measuring a first magnetic signal value for said hybridized doublestrand; d) incubating a third nucleotide strand with said hybridizeddouble strand; wherein said third strand is complementary to said firststrand, and wherein said incubating forms an exchange product; e)measuring a second magnetic signal value for the exchange product ofstep d after applying a weak mechanical force to remove nonspecificallybound magnetic particles; and f) quantifying the amount of said thirdnucleotide strand from the difference in magnetic signal values measuredin step c and step e, wherein the difference in the magnetic signalvalues comprises a decrease in magnetic signal values due to therandomization of the second nucleotide single strand magnetic dipoles.2. The method of claim 1, wherein said first nucleotide single strand isderivatized.
 3. The method of claim 1, wherein said first nucleotidestrand is immobilized to said surface through a S—Au covalent bond or bya streptavidin-biotin covalent bond.
 4. The method of claim 1, whereinsaid magnetic particle is about 1 nm to about 10 μm in size.
 5. Themethod of claim 1, wherein said first and said second magnetic signalcomprise magnetic moment measurements.
 6. The method of claim 5, whereinstep f comprises measuring the change in magnetic signal (ΔB),optionally the method further comprising calculating the molarconcentration of said third nucleotide strand, wherein saidconcentration is linearly related to ΔB.
 7. The method of claim 1,wherein said quantifying further comprises calculating the number offree magnetic particle labels, wherein the number of said free magneticparticles corresponds to the number of exchange product molecules. 8.The method of claim 1, wherein said weak mechanical force is supplied bya shaker, centrifuge, or sonicator.
 9. The method of claim 1, whereinsaid hybridized strand is in a liquid environment, a cell lysate, bloodplasma, or urine.
 10. The method of claim 1, wherein said firstnucleotide strand is a RNA or a DNA sequence of about 1-100 nucleotides.11. The method of claim 1, wherein said third nucleotide strand is a DNAor microRNA sequence of about 1 to about 100 nucleotides.
 12. The methodof claim 1, wherein said exchange product is thermodynamically morestable than said hybridized double strand.
 13. A method ofsimultaneously detecting an array of heterologous nucleotide sequences;the method comprising: coating a sample well comprising an array ofcompartments; wherein the surface of said compartments arealternatively: a) coated with hybridized nucleotide double strandcomprising a strand comprising a magnetic particle; and b) uncoated;wherein said uncoated compartment produces no magnetic signal; and eachsaid coated compartment comprises a heterologous hybridized doublestrand sequence; c) measuring magnetic signals for each compartment; d)incubating said array with a sample comprising free target nucleotidesequences, and forming exchange products; e) measuring magnetic signalsfor each compartment comprising exchange products; f) calculating thedifference in said signals from step c and e wherein the difference insaid signals comprises a decrease in magnetic signals due to therandomization of magnetic dipoles; and g) quantifying and identifyingsaid target sequence based on the change in signal calculated in f. 14.An exchange induced remnant magnetization method to detect cancerbiomarkers, the method comprising: (a) immobilizing a first sequence ona surface; wherein said first sequence comprises N bases; (b) adding asecond sequence to said first sequence, wherein said second sequencecomprises less than N complementary bases and a magnetic particle; andwherein said second sequence hybridizes to said first sequence forming ahybridized double strand; and (c) measuring a first magnetic signalvalue for said hybridized double strand; (d) incubating said hybridizeddouble strand with a biomarker nucleic acid; and wherein said biomarkerexchanges with said second strand to form an exchange product; whereinsaid exchange product is thermodynamically more stable than saidhybridized double strand; (e) measuring a second magnetic signal valuefor the exchange product of step (c) after applying a weak mechanicalforce to remove nonspecifically bound magnetic particles; and (f)quantifying the amount of said biomarker from the difference in magneticsignal values measured in step (c) and step (e) wherein the differencein the magnetic signal values comprises a decrease in magnetic signalvalues due to the randomization of the second sequence magnetic dipoles.